<p>In clinical practice, the measurement of parathyroid hormone (PTH) typically employs immunoassays, but these methods lack comparability of results due to variations in specificity or calibration. Consequently, there is a clear need to establish a highly accurate and comparable procedure for a PTH calibrator. Purity assessment for a high-purity material is a prerequisite for the standardization of PTH assay. The present study proposes an accurate quantitative method for PTH purity in high-purity materials, integrating amino acid (AA) and peptide analysis for quantitative purposes. In the beginning, a liquid chromatography mass spectrometry (LC–MS) analysis was performed to confirm its molecular weight and AA sequence. Subsequently, three quantitative AAs were selected for accurate quantification of PTH by amino acid isotope dilution mass spectrometry (AA-IDMS). Finally, in peptide-IDMS, peptide ADVNVLTK (AK) was selected as the quantitative peptide to quantify PTH. The results of the two IDMS quantification methods exhibited strong concordance, with both exhibiting minimal uncertainty. The final mass fraction of PTH in the study material was calculated to be 0.718 ± 0.022&#xa0;g/g. The method developed in this study provides novel technical support and theoretical foundations for the quality assessment and quality standard formulation of PTH-related products. It has great application potential in the purity determination of other bioactive substances with similar properties as PTH, and would promote the development of the field of bioanalysis.</p> Graphical abstract <p></p>